In order to identify scan coordinate values (φn, θn) for operating a scanning unit (28) of a confocal scanning microscope (20), spherical scan coordinate values (φn, θn) are identified, as a function of cartesian image coordinates (Xn, Yn) of image points of an image (60) to be created of a sample (32), with the aid of a coordinate transformation of the cartesian image coordinate values (Xn, Yn) into a spherical coordinate system. The scanning unit (28) is operated as a function of the spherical scan coordinate values (φn, θn).
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1. A method for identifying scan coordinate values for operating a scanning unit of a confocal scanning microscope, the method comprising:
directing an illumination light beam via the scanning unit of the scanning microscope onto a sample;
identifying spherical scan coordinate values as a function of cartesian image coordinates of image points of an image to be created of the sample, with the aid of a coordinate transformation of the cartesian image coordinate values into a spherical coordinate system;
considering different possible zoom steps of the scanning microscope, by predefining spacings of the image points with respect to one another as a function of the zoom steps, when the spherical scan coordinate values are identified; and
operating the scanning unit as a function of the spherical scan coordinate values.
8. A method for operating a scanning unit of a confocal scanning microscope, comprising:
identifying spherical scan coordinate values as a function of cartesian image coordinate values of image points of an image to be created of a sample, with the aid of a coordinate transformation of the cartesian image coordinate values into corresponding spherical scan coordinate values of a spherical coordinate system;
storing the cartesian image coordinate values and the corresponding spherical scan coordinate values in an association specification;
considering different possible zoom steps of the microscope, by predefining spacings of the image points with respect to one another as a function of the zoom steps, when the spherical scan coordinate values are identified; and
operating the scanning unit as a function of the stored spherical scan coordinate values.
7. A method for creating an image of a sample with the aid of a confocal scanning microscope, the method comprising:
directing a illumination light beam onto a sample via a scanning unit of the scanning microscope;
driving the scanning unit, as a function of predefined scan coordinate values, in such a way that the illumination light beam optically scans the sample;
identifying scan coordinate values of the scanning unit that are representative of true positions of the scanning unit;
sensing detected light proceeding from the sample as a function of the identified scan coordinate values;
correcting the identified scan coordinate values;
associating the corrected scan coordinate values with image coordinate values of image points of an image of the sample that are to be identified;
identifying the image points at the image coordinate values on the basis of the detected light sensed at the corresponding corrected scan coordinate values;
considering different possible zoom steps of the scanning microscope, by predefining spacings of the image points with respect to one another as a function of the zoom steps, when the image coordinate values are identified; and
creating the image of the sample on the basis of the identified image points.
4. A method for identifying corrected scan coordinate values for operating a scanning unit of a confocal scanning microscope, the method comprising:
directing an illumination light beam via the scanning unit of the scanning microscope onto a reference sample;
driving the scanning unit, as a function of predefined scan coordinate values, in such a way that the illumination light beam optically scans the reference sample;
sending detected light proceeding from the reference sample as a function of the predefined scan coordinate values;
associating the predefined scan coordinate values with image coordinate values of image points of an image to be identified of the reference sample;
identifying the image points at the image coordinate values on the basis of the detected light sensed at the corresponding predefined scan coordinate values;
creating the image of the reference sample on the basis of the identified image points;
comparing the image of the reference sample with the reference sample; and
correcting the predefined scan coordinate values as a function of the comparison,
wherein different possible zoom steps of the scanning microscope are considered, by predefining spacings of the image points with respect to one another as a function of the zoom steps, when the image coordinate values identify the image points.
6. A method for creating an image of a sample with the aid of a confocal scanning microscope, the method comprising:
directing an illumination light beam onto a sample via a scanning unit of the scanning microscope;
identifying spherical scan coordinate values as a function of cartesian image coordinate values of image points of the image to be created of the sample by transforming the cartesian image coordinate values into a spherical coordinate system;
storing the cartesian image coordinate values and the corresponding spherical scan coordinate values in an association specification table;
driving the scanning unit, as a function of the stored spherical scan coordinate values, in such a way that the illumination light beam optically scans the sample;
identifying spherical scan coordinate values of the scanning unit that are representative of true positions of the scanning unit;
correcting the identified spherical scan coordinate values;
sensing detected light proceeding from the sample as a function of the corrected spherical scan coordinate values;
associating the corrected spherical scan coordinate values with the stored spherical image coordinate values of the image points of the image of the sample that are to be identified;
identifying the image points on the basis of the detected light sensed at the corresponding corrected spherical scan coordinate values;
considering different possible zoom steps of the scanning microscope, by predefining spacings of the image points with respect to one another as a function of the zoom steps, when the spherical scan coordinate values are identified; and
creating the image of the sample on the basis of the identified image points.
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This application claims priority to German Patent Application No. DE 10 2010 061 612.5, filed Dec. 29, 2010, which is incorporated herein by reference in its entirety.
The invention relates to a method for identifying scan coordinate values or corrected scan coordinate values for operating a scanning unit of a confocal scanning microscope. The invention further relates to a method for creating an image of a sample with the aid of a confocal scanning microscope.
A confocal scanning microscope can be used, for example, to excite fluorescing substances in samples, in particular tissue samples, and to detect the fluorescent light resulting therefrom. The principle is that an illumination light beam is directed via a scanning unit onto a sample, the scanning unit being operated with a driving signal that encompasses scan coordinate values. The scan coordinate values are representative of positions of positioning elements of the scanning unit. The fluorescent light radiated from the sample is directed, via the scanning unit and a beam splitter that separates the fluorescent light from the illumination light, onto a detection pinhole that allows a small portion of the detected light to pass through to a detector. The detected light sensed with the aid of the detector is detected as a function of the scan coordinate values. In other words, in addition to the signal detected by the scanning unit, the position occupied by the positioning elements of the scanning unit upon detection of the detected light is also recorded, the positions being represented by the scan coordinate values.
The detected light can be detected as a function of the scan coordinate values with which the scanning unit is driven; if the scanning unit (in particular its positions) is controlled in closed-loop fashion, the predefined scan coordinate values can also be referred to as “target” scan coordinate values. Alternatively thereto, true positions of the scanning unit can be sensed with the aid of sensors, and these sensed scan coordinate values can then be associated with the detected light detected at the sensed positions; once again, if the positions of the scanning unit are controlled in closed-loop fashion, these sensed scan coordinate values can also be referred to as “actual” scan coordinate values.
During optical scanning of the sample or thereafter, the image of the sample can be assembled on the basis of many acquired image points, the position of each image point being determined by a pair of image coordinate values, and corresponding scan coordinate values being associated with the image coordinate values. It is known to determine this association of the image coordinate values with the scan coordinate values by means of approximations. This can result, however, in distortions and/or deformations, in particular at the edges of the image of the sample.
The distortions of the images occur not only because of the approximately calculated scan coordinate values, but also because of optical effects of the optical elements of the confocal scanning microscope which are not considered in known scanning microscopes or methods for operating the scanning microscopes.
The distortions are problematic in particular when an overall image of a sample is to be assembled from multiple juxtaposed individual images, since the images then do not match one another at their edges. This can furthermore be problematic when the image of the sample, acquired confocally in this fashion, is to be overlaid on a wide field image, since such distortions at the image edges do not occur with wide field imaging and the two images of the sample thus do not fit together, especially at the edges of the images.
It is an object of the present invention to establish a method for identifying scan coordinate values or corrected scan coordinate values for operating a scanning unit of a confocal scanning microscope, or a method for creating an image of a sample with the aid of a scanning microscope, which enable the creation of a precise image of the sample, in particular at the image edges, using simple means.
The object is achieved by the features of the independent claims. Advantageous embodiments are indicated in the dependent claims.
According to a first aspect, the invention is characterized by the fact that spherical scan coordinate values are identified, as a function of Cartesian image coordinates of image points of an image to be created of a sample, with the aid of a coordinate transformation of the Cartesian image coordinate values into a spherical coordinate system. The scanning unit is operated as a function of the spherical scan coordinate values.
Transformation of the Cartesian image coordinate values into the spherical coordinate system contributes to a precise association of the image points of the image of the sample, which were identified by detection of the detected light, with positions of the scanning unit of the confocal scanning microscope, the positions of the scanning unit being predefined by the spherical scan coordinate values. The result of this is that the image of the sample corresponds precisely to the sample, in particular at the edges of the image.
The spherical scan coordinate values exist in a format, or are formatted, such that the scanning unit is operable as a function of the spherical scan coordinate values. This means in this connection, for example, that the spherical scan coordinate values are saved in an association specification, for example a table, that is then stored on a memory unit of a control unit of the scanning microscope and is read out by the control unit during operation of the confocal scanning microscope. A driving signal is modulated in such a way that positioning elements of the scanning unit assume positions that correspond to the spherical scan coordinate values. Alternatively thereto, the spherical scan coordinate values can be formatted in exactly the way in which they already exist (without transformation) as a driving signal, for example modulated therein; and these data are stored, for example in digitized fashion, on the memory unit of the control unit. The positions of the scanning unit are referred in each case to angles enclosed between an illumination light beam deflected with the aid of the scanning unit, and a reference light beam deflected with the aid of the scanning unit in a reference position of the scanning unit.
In an advantageous embodiment, different zoom steps of the scanning microscope are considered when the spherical scan coordinate values are identified. “Zoom” means a limitation of a region or of a coordinate region. The zoom steps correspond to magnification steps, and can also be referred to as such. It is particularly advantageous in this connection if the different zoom steps are considered by predefining spacings of the image points with respect to one another as a function of the zoom steps. Consideration of the zoom steps makes it possible always to ensure the same precision in image creation upon later operation of the scanning microscope, regardless of the zoom step.
According to a refinement, only first spherical scan coordinate values are identified with the aid of the coordinate transformation, and second spherical scan coordinate values are identified by interpolation of the first scan coordinate values. This is advantageous in particular when only limited computing performance is available, or when identification of the spherical scan coordinate values must be carried out particularly quickly, for example during operation of the scanning microscope.
A refinement provides that the predefined scan coordinate values are corrected on the basis of the spherical scan coordinate values. This allows an already existing scanning microscope, in particular its scanning unit, to be driven in such a way that precise identification of the images is possible. The predefined scan coordinate values can, for example, be replaced by the spherical scan coordinate values; or only differences between the predefined scan coordinate values and the spherical scan coordinate values can be identified, and the predefined scan coordinate values are corrected on the basis only of the differences, which in this connection can also be referred to as “correction terms.”
According to a second aspect, the invention is notable for the fact that an illumination light beam is directed via a scanning unit of the scanning microscope onto a reference sample. The scanning unit is driven, as a function of predefined scan coordinate values, in such a way that the illumination light beam optically scans the reference sample. Detected light proceeding from the reference sample is sensed as a function of the predefined scan coordinate values. The predefined scan coordinate values are associated with image coordinate values of image points of an image to be identified of the reference sample. The image points at the image coordinate values are identified on the basis of the detected light sensed at the corresponding predefined scan coordinate values. The image of the reference sample is created on the basis of the identified image points. The image of the reference sample is compared with the reference sample, and the predefined scan coordinate values are corrected as a function of the comparison.
Creation of the image of the reference sample, and correction of the predefined scan coordinate values on the basis of the image created, can contribute, for example, to the ability to identify, in a context of relatively imprecise predefined scan coordinate values, correction terms with which the relatively imprecise scan coordinate values can be replaced with scan coordinate values that are relatively precise with respect thereto. Without correction with the aid of the reference sample, it is possible for distortions of the image of the sample to occur as a result of optical effects of the scanning microscope even when precise predefined scan coordinates (such as, for example, the spherical scan coordinate values recited above) exist. These distortions can be considered and eliminated with the aid of the reference sample.
Any sample that has a well-defined structure, for example a closely-spaced lattice, is suitable as a reference sample. A reference sample having a very precise reference pattern can be generated, for example, by etching a semiconductor substrate, in a manner known from microchip manufacture. The comparison between the image of the reference sample and the reference sample can occur, for example, by comparing the image of the reference sample with a precisely generated reference image of the reference sample. The comparison can, in particular, be carried out automatically with the aid of an image processing system on a computer. Correction of the predefined scan coordinate values can occur, for example, by replacing the predefined scan coordinate values with the corrected scan coordinate values; or only the difference values between the predefined scan coordinate values and the corrected scan coordinate values can be identified, and the differences thus identified, constituting correction terms, can be correlated with the predefined scan coordinate values.
In a refinement, the image of the reference sample is acquired at different zoom steps of the scanning microscope, and the predefined scan coordinate values are corrected as a function of the corresponding zoom step. In other words, the method for identifying corrected scan coordinate values is carried out at different magnifications of the scanning microscope, and the identified corrected scan coordinate values, or the corresponding correction terms, are saved in a manner associated with the corresponding zoom or magnification steps, the scan coordinate values being predefined later on, during operation of the scanning microscope, in accordance with the zoom step that is currently set. The method for identifying the corrected scan coordinate values can be carried out before the confocal scanning microscope is used as intended, or also after the scanning microscope is completed, for example in the form of a regular calibration.
According to a third aspect, the invention is notable for the fact that the illumination light beam is directed onto a sample via the scanning unit of the scanning microscope. The scanning unit is driven, as a function of predefined scan coordinate values, in such a way that the illumination light beam optically scans the sample. Scan coordinate values of the scanning unit that are representative of true positions of the scanning unit are identified. The identified scan coordinate values are corrected. Detected light proceeding from the sample is sensed as a function of the corrected scan coordinate values. The corrected scan coordinate values are associated with image coordinate values in order to identify the image points of an image of the sample. The image points at the image coordinates are identified on the basis of the detected light sensed at the corresponding corrected scan coordinate values. The image of the sample is created on the basis of the identified image points.
According to a fourth aspect, the invention is notable for a method that largely corresponds to the method according to the third aspect; in contrast thereto, the detected light proceeding from the sample is first sensed as a function of the identified scan coordinate values, and then the identified scan coordinate values are corrected, the result being that the association of the detected light with the scan coordinate values is modified.
According to the first or second aspect, the predefined and/or spherical and/or corrected scan coordinate values can in principle be used to drive the scanning unit. Correction of the identified scan coordinate values in accordance with aspects three and four refers, in contrast thereto, to scan coordinate values that are sensed during operation of the scanning microscope. If the scanning unit, and in particular the positioning elements of the scanning unit, are operated with a closed-loop control system, the predefined scan coordinate values can then also be referred to as “target” scan coordinate values, and the identified scan coordinate value also as “actual” scan coordinate values. In other words, for example, in accordance with the first and the second aspect a correction of the target scan coordinate values can occur; and in accordance with the third or fourth aspect a correction of actual scan coordinate values can occur. In this connection, for example, the predefined scan coordinate values can be the aforementioned spherical scan coordinate values or the corrected scan coordinate values, and/or the identified scan coordinate values can be the uncorrected scan coordinate values.
Exemplifying embodiments of the invention are further explained below with reference to schematic drawings, in which
Elements of identical design or function are labeled with the same reference characters throughout the Figures.
The sample is, for example, a tissue sample which comprises fluorescing substances that, for example, were introduced into the sample, and/or which comprises substances that are activatable with the aid of illumination light beam 24 and, in the active state, can be excited to fluoresce.
Any sample that has a well-defined structure, for example a sample that comprises a closely-spaced lattice, is suitable as reference sample 32. A reference sample 32 of this kind can be etched out of a semiconductor substrate, for example as in the manufacture of a microchip. This can contribute to extremely precise manufacture of the structures of the reference sample, even in the sub-nanometer range.
The detected light proceeding from the sample accordingly encompasses, for example, fluorescent light or reflected light that, in the form of a detected light beam 34, passes through objective 30 to scanning unit 28, which directs detected light beam 34 onto main beam splitter 26, which allows the detected light to pass through to a detection pinhole 36, for example an aperture, to a detector 38.
Scanning unit 28 is operated with scan coordinate values stored on a memory medium of the control unit, and the sensed detected light is sensed as a function of the scan coordinate values. In other words, with the aid of the control unit the sensed detected light is associated with the positions of scanning unit 28 that are currently set, which are represented by the scan coordinate values. These scan coordinate values can be the scan coordinate values with which scanning unit 28 is driven, or, alternatively thereto, sensors can be provided which sense the true positions of the positioning units of scanning unit 28; the scan coordinate values sensed in that fashion can then be associated with the correspondingly sensed detected light. If the positioning elements of scanning unit 28 are operated with the aid of a closed-loop control system, the scan coordinate values with which scanning unit 28 is driven can also be referred to as “target” scan coordinate values, and the sensed scan coordinate values can be referred to as “actual” scan coordinate values.
A transformation arrow 50 is representative of an illumination light beam 24 deflected with respect to the reference light beam. Transformation arrow 50 is at an angle φ1 to Z axis 40, and at an angle Φ1 to the plane spanned by X axis 40 and Z axis 44. Illumination light beam 24, deflected in this fashion, arrives in intermediate image plane 52 at a point x1, y1 that corresponds in sample plane 54 to a point on the sample having coordinate values X1, Y1. The angles φr and θn can also be referred to as “scan coordinate values.”
Formula F1 represents the principle of the transformation of Cartesian image coordinates into a spherical coordinate system. Formula F2 represents the transformation with the aid of transformation arrow 50. The transformation arrow refers to the “r(θ;φ)” in formula F2, where r0 is the distance between the point around which the beam is deflected and intermediate image plane 52 (referring to
The spherical coordinate values φn, θn, are obtained preferably with the aid of the coordinate transformation explained in
In a step S2, reference sample 32 is optically scanned as a function of predefined scan coordinate values. The predefined scan coordinate values can be the spherical scan coordinate values φn, θn or scan coordinate values that are imprecise relative thereto.
In a step S4, the detected light reflected from reference sample 32 is detected as a function of the predefined scan coordinate values. In other words, what is sensed is, inter alia, how scanning unit 28 is being activated at the moment when a specific component of the detected light is sensed.
In a step S6, image 60 of reference sample 32 is created as a function of the detected light. In particular, the image of reference sample 32 is assembled from individual image points, and the image points are identified on the basis of the detected light. What can occur here is association of the corresponding predefined scan coordinate values, in particular the spherical scan coordinate values φn, θn or the corrected scan coordinate values φnK, θnK corrected with the aid of the spherical scan coordinate values φn, θn, with the Cartesian image coordinate values Xn, Yn, of the image points, with the aid of the association specification shown in
In a step S8, image 60 of reference sample 32 is compared with reference sample 32. For example, a reference image of reference sample 32 can be created, and image 60 of reference sample 32 can be compared with the reference image, for example with the aid of an image processing program that is stored on a computer. The comparison encompasses the detection of deviations, in particular distortions, between image 60 of reference sample 32 and the reference image. For example, intersection points of image 60 can be compared with intersection points of the reference image, and their spacing and displacement direction can be identified and used for correction.
In a step S10, the predefined scan coordinate values are corrected as a function of the comparison. In this correction, in particular, the displacement of the reference points is taken into consideration in such a way that upon subsequent driving of scanning unit 28, the intersection points of image 60 correspond to the reference points in reference sample 32. The corrected scan coordinate values φnK, θnK can be stored in the association specification, in addition to the spherical scan coordinate values φn, θn or the predefined scan coordinate values. Alternatively thereto, the predefined scan coordinate values can be replaced by the corrected ones φnK, θnK. Alternatively thereto, only the difference terms between the predefined scan coordinate values and the corrected scan coordinate values φnK, θnK can be identified, and stored in correlation with the predefined scan coordinate values.
When further samples are examined, for example tissue samples, scanning unit 28 can then, as described in connection with
In a step S14, scan coordinate values that correspond to the true positions of the positioning elements of scanning unit 28 are sensed. It is preferable to provide for this purpose sensors that sense the positions of the positioning elements. If scanning unit 28 is operated with the aid of the closed-loop control system, the sensed scan coordinate values can also be referred to as “actual” scan coordinate values.
In a step S16, the sensed scan coordinate values are corrected. By preference, the sensed scan coordinate values (if they are the scan coordinate values determined by approximation) are corrected with the aid of the spherical scan coordinate values φn, θn and/or with the aid of the correction shown by the method in
In a step 18, the detected light is sensed as a function of the corrected scan coordinate values.
In a step S20, the image of the sample is assembled on the basis of the sensed detected light, corresponding to creation of the image of reference sample 32 in step S6 of the method shown in
In other words, in the method shown in
The confocal scanning microscope 20 shown in
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